1887

Abstract

The flavin adenine dinucleotide (FAD)-containing putrescine oxidase of catalyses the oxidative deamination of putrescine. The amino acid sequences of the NH-termini of the mature enzyme and lysyl-endopeptidase-generated fragments were determined for preparation of synthetic oligonucleotides as hybridization probes for cloning. A 4·4 kb HI fragment which contained DNA sequences hybridizing to the probes was cloned in pUC19 in . The nucleotide sequence together with the determined amino acid sequences revealed that this enzyme consists of 480 amino acids ( 52000) and contains an FAD-binding consensus sequence at its NH-terminal portion. In front of the transcriptional start point, which is 28 bases upstream of the initiation codon as determined by primer extension, −35 and −10 sequences similar to typical prokaryotic promoter consensus sequences are present. JM109 containing the putrescine oxidase gene just downstream of the promoter in pUC18 produced a large amount of this protein when grown at 37 °C but in the enzymically inactive form of inclusion bodies. However, cultivation of the recombinant cells at temperatures below 30°C led to production of active enzyme (20 times as much as produced by the original strain).

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1993-03-01
2021-10-20
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